human tgfβ2  (PeproTech)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Small Extracellular Vesicle Treatment of Trabecular Meshwork Fibrosis: 2D/3D In Vitro and In Vivo Analyses

doi: 10.1167/iovs.66.12.48

Figure Lengend Snippet: Effects of anterior chamber injection of hBMMSC sEVs on IOP reduction and antifibrotic activity in mice with ocular hypertension. ( A ) IOP trends over 28 days among three groups of mice ( n = 8). *Significant differences in IOP between the antifibrotic group and the fibrotic group. #Significant differences between the fibrotic and control groups. ( B ) Autofluorescence of Ad-TGFβ2 C226/228S in the anterior chamber angle observed in frozen sections using confocal microscopy. (The TM region is demarcated by yellow dashed circles .) ( C – E ) Immunofluorescence analysis of FN and α-SMA expression in the anterior chamber angle ( n = 8). The y axis represents the mean fluorescence integrated density per unit area ( IntDen/Area ) in the TM region (demarcated by white dashed circles ). Statistical significance: * P < 0.05; *** P < 0.001; **** P < 0.0001; ### P < 0.001; ns, not significant.

Article Snippet: The fibrotic cells were treated with 5 ng/mL, 10 ng/mL, or 20 ng/mL TGFβ2 (HY-P7119, MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Injection, Activity Assay, Control, Confocal Microscopy, Immunofluorescence, Expressing, Fluorescence

Journal: Fluids and Barriers of the CNS

Article Title: Exploring dysfunctional barrier phenotypes associated with glaucoma using a human pluripotent stem cell-based model of the neurovascular unit

doi: 10.1186/s12987-024-00593-x

Figure Lengend Snippet: Identification of TGFβ2 as a candidate molecule to modulate barrier integrity. ( A ) A specific and significant increase in the transcriptional expression of TGFβ2 in OPTN(E50K) astrocytes compared to isogenic control astrocytes was observed by analyzing RNA-seq data (GSE173129), while no significant differences were observed in the expression of TGFβ1 or TGFβ3. Student’s t-test determined *** p < 0.001. ( B ) At the protein level, OPTN(E50K) astrocytes secreted increased levels of TGFβ2 compared to isogenic control astrocytes, as determined by ELISA. Student’s t-test determined ** p = 0.0026. ( C ) Schematic demonstrating the experimental culture system consisting of isogenic control RGCs and astrocytes cocultured with MVECs in the transwell insert model. The exogenous addition of TGFβ2 to triple cocultures of MVECs with isogenic control RGCs and astrocytes resulted in a significantly decreased TEER ( D ) and increased sodium fluorescein permeability ( E ), mimicking trends observed for OPTN(E50K) RGCs and astrocytes. ( F ) Schematic demonstrating the experimental culture system consisting of MVECs co-cultured with OPTN(E50K) astrocytes and RGCs. The exogenous addition of TGFβ neutralizing antibodies led to a significant increase in TEER ( G ) as well as a significant decreased in sodium fluorescein permeability ( H ) in triple co-cultures of MVECs with OPTN(E50K) RGCs and astrocytes, mimicking trends observed for isogenic control RGCs and astrocytes. Data represents mean values ± SEM from at least five independent differentiation experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001, one-way ANOVA followed by Tukey’s multiple comparison test in A , D , E , G and H

Article Snippet: To stimulate isogenic control co-cultures, TGFβ2 (R&D Systems, 302-B2) was added to barrier models at a final concentration of 10 ng/ml.

Techniques: Expressing, Control, RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Permeability, Cell Culture, Comparison

Journal: MedComm

Article Title: Transforming Growth Factor Beta2 Promotes Migration and Inhibits the Proliferation of Gastric Cancer Cells by Regulating the pSmad2/3‐NDRG1 Signaling Pathway

doi: 10.1002/mco2.70148

Figure Lengend Snippet: The expression levels of TGFβ2 in gastric cancer tissues and normal tissues were analyzed using data from the TCGA and GTEx databases (A). The relationship between TGFβ2 expression levels and overall survival time was analyzed (B). Representative images of IHC staining in gastric cancer tissues and adjacent noncancerous tissues, using an antibody against TGFβ2, are shown (C). In 212 paired samples, TGFβ2 expression was significantly different between gastric cancer tissues and adjacent noncancerous tissues (D). The effects of stable TGFβ2 interference and overexpression in gastric cancer cell lines were confirmed by Western blot analysis (E). Analysis of gastric cancer samples revealed a significant association between upregulated TGFβ2 expression and 5‐year overall survival in patients with gastric cancer (F). TGFβ2 interference and overexpression were validated at the RNA level (G) (scale bars: 500 and 50 µm). Statistical significance is indicated as follows: ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human TGFβ2 protein (HZ‐1092, Proteintech) was dissolved in sterile water and the mixture was added to the cell culture dishes in gradient concentrations to achieve final concentrations of 0, 5, 10, 15, and 20 ng/mL.

Techniques: Expressing, Immunohistochemistry, Over Expression, Western Blot

Journal: MedComm

Article Title: Transforming Growth Factor Beta2 Promotes Migration and Inhibits the Proliferation of Gastric Cancer Cells by Regulating the pSmad2/3‐NDRG1 Signaling Pathway

doi: 10.1002/mco2.70148

Figure Lengend Snippet: Migration assays of AGS and MGC803 cells with TGFβ2 interference or overexpression were performed (A), and corresponding statistical analyses are shown on the right side (B). The metastatic ability of TGFβ2‐overexpressing MGC803 cells was assessed by in vivo imaging using D‐Luciferin (C), and the corresponding quantitative analysis of fluorescence intensity is shown on the right side (D). TGFβ2 expression was immunohistochemically detected in lung metastatic tumors from mice (E) (scale bars: 500 and 50 µm). The proliferation rates of AGS and MGC803 cells in different treatment groups were measured using the CCK‐8 assay (F). The colony‐formation ability of AGS and MGC803 cells was compared using a plate colony assay in different treatment groups (G), and the corresponding statistical analyses are shown on the right side (H). Statistical significance is indicated as follows: * p < 0.05, *** p < 0.001.

Article Snippet: Recombinant human TGFβ2 protein (HZ‐1092, Proteintech) was dissolved in sterile water and the mixture was added to the cell culture dishes in gradient concentrations to achieve final concentrations of 0, 5, 10, 15, and 20 ng/mL.

Techniques: Migration, Over Expression, In Vivo Imaging, Fluorescence, Expressing, CCK-8 Assay, Colony Assay

Journal: MedComm

Article Title: Transforming Growth Factor Beta2 Promotes Migration and Inhibits the Proliferation of Gastric Cancer Cells by Regulating the pSmad2/3‐NDRG1 Signaling Pathway

doi: 10.1002/mco2.70148

Figure Lengend Snippet: The heatmap shows the top 30 genes with significant expression changes identified by RNA sequencing following TGFβ2 overexpression (A). Western blotting was performed to analyze TGFβ2 and NDRG1 expression in AGS and MGC803 cells, with GAPDH as the loading control (B, D). The mRNA levels of NDRG1 in AGS and MGC803 cells were measured by real‐time qPCR, with GAPDH as the loading control (C, E). NDRG1 (orange) was detected in AGS and MGC803 cells by immunofluorescence staining (F). NDRG1 expression in AGS and MGC803 cells was analyzed by Western blotting and real‐time qPCR, with GAPDH as the loading control (G, H). NDRG1 expression in AGS and MGC803 cells was further confirmed by Western blotting and real‐time qPCR, with GAPDH as the loading control (I–K). Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human TGFβ2 protein (HZ‐1092, Proteintech) was dissolved in sterile water and the mixture was added to the cell culture dishes in gradient concentrations to achieve final concentrations of 0, 5, 10, 15, and 20 ng/mL.

Techniques: Expressing, RNA Sequencing, Over Expression, Western Blot, Control, Immunofluorescence, Staining

Journal: MedComm

Article Title: Transforming Growth Factor Beta2 Promotes Migration and Inhibits the Proliferation of Gastric Cancer Cells by Regulating the pSmad2/3‐NDRG1 Signaling Pathway

doi: 10.1002/mco2.70148

Figure Lengend Snippet: The lung metastatic ability of NDRG1‐overexpressing MGC803 cells was measured by in vivo imaging using D‐Luciferin, and the corresponding quantitative analysis of fluorescence intensity is shown on the right side (A). NDRG1 expression was immunohistochemically detected in lung metastatic tumors from mice (B). The migration assay of AGS and MGC803 cells with NDRG1 expression interference, cocultured with exogenous recombinant TGFβ2 protein, is displayed on the right side (C, D), along with corresponding statistical analyses. As the concentration of exogenous recombinant human TGFβ2 increased, the inhibition of GC cell proliferation became more significant (E). Overexpression of TGFβ2 promoted cell migration and upregulated NDRG1 expression, which was reversed by NDRG1 knockdown (F, G) (scale bars: 500 and 50 µm). Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human TGFβ2 protein (HZ‐1092, Proteintech) was dissolved in sterile water and the mixture was added to the cell culture dishes in gradient concentrations to achieve final concentrations of 0, 5, 10, 15, and 20 ng/mL.

Techniques: In Vivo Imaging, Fluorescence, Expressing, Migration, Recombinant, Concentration Assay, Inhibition, Over Expression, Knockdown

Journal: MedComm

Article Title: Transforming Growth Factor Beta2 Promotes Migration and Inhibits the Proliferation of Gastric Cancer Cells by Regulating the pSmad2/3‐NDRG1 Signaling Pathway

doi: 10.1002/mco2.70148

Figure Lengend Snippet: Western blotting of TGFβ2, Smad2/3, pSmad2/3, E‐cadherin, and N‐cadherin expression in AGS and MGC803 cells with TGFβ2 interference or overexpression. GAPDH served as the internal control (A). Effects of SIS3 concentration on NDRG1 expression in AGS and MGC803 cells (B). Effect of TGFβ /Smad inhibitor Trabedersen on NDRG1 expression in MGC803 cells (C). Effect of siRNA interference on Smad2/3 expression (D). Interference of Smad2, Smad3, and SMAD2/3 expression on NDRG1 protein and RNA level in AGS and MGC803 cells (E, F). PCR‐amplified products from ChIP assays of MGC803 cells were detected by Southern blotting. Input acted as a positive control, while IgG served as a negative control (G, H). Statistical significance is indicated as follows: * p < 0.05, *** p < 0.001, ns: not significant.

Article Snippet: Recombinant human TGFβ2 protein (HZ‐1092, Proteintech) was dissolved in sterile water and the mixture was added to the cell culture dishes in gradient concentrations to achieve final concentrations of 0, 5, 10, 15, and 20 ng/mL.

Techniques: Western Blot, Expressing, Over Expression, Control, Concentration Assay, Amplification, Southern Blot, Positive Control, Negative Control

Journal: MedComm

Article Title: Transforming Growth Factor Beta2 Promotes Migration and Inhibits the Proliferation of Gastric Cancer Cells by Regulating the pSmad2/3‐NDRG1 Signaling Pathway

doi: 10.1002/mco2.70148

Figure Lengend Snippet: Migration assays of AGS and MGC803 cells following the interference of Smad2, Smad3, or SMAD2/3 expression, respectively, with corresponding statistical analyses shown on the right side (A−C) (scale bars: 100 µm). Western blotting of Smad2/3, pSmad2/3, and NDRG1 expression in AGS and MGC803 cells (D, E). Representative IHC staining for NDRG1 in gastric cancer samples (F) with corresponding statistical analyses (G) shown on the right side (scale bars: 100 and 500 µm). Spearman correlation analysis of IHC staining scores of TGFβ2 and NDRG1: r = 0.74, p < 0.001 (H). Relationship between NDRG1 expression levels in gastric cancer tissues and patient OS (I): p = 0.0027. Statistical significance is indicated as follows: ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: Recombinant human TGFβ2 protein (HZ‐1092, Proteintech) was dissolved in sterile water and the mixture was added to the cell culture dishes in gradient concentrations to achieve final concentrations of 0, 5, 10, 15, and 20 ng/mL.

Techniques: Migration, Expressing, Western Blot, Immunohistochemistry

Journal: bioRxiv

Article Title: TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes

doi: 10.1101/2024.11.05.622187

Figure Lengend Snippet: ( A ) TGFβ2 treatments for 24 hours at 1 ng/mL (N = 6 pTM strains, n = 3 - 5 slides/condition/day) or 5 ng/mL (N = 5 pTM strains, n = 3 - 5 slides/condition/day) did not show potentiation of GSK101-evoked TRPV4 Ca 2+ influx (SI Appendix, Figure S1) and were significantly lower than cells treated with TGFβ2 for 5d at 1ng/mL (5d TGFβ2 results from ). Individual data points over mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test, statistics for individual 1d treatment groups compared to control groups shown in Figure S1. ( B ) Representative traces for GSK101 response following 24-hour TGFβ2 treatment, traces show mean ± SEM of 3-4 cells. ( C ) Average current density in response to GSK101 (24-hour control: n = 11 cells, 24-hour TGFβ2: n=10 cells) shows generally increased current in TGFβ2-treated cells. Data shows mean ± SEM ( D - E ) Violin plots of individual cell strains shown in A . Thick dashed line indicates mean, while light dashed line indicates quartiles. ** P < 0.01, *** P < 0.001

Article Snippet: Recombinant human TGFβ2 protein was ordered from R&D Systems and reconstituted in sterile 4 mM HCl with 0.1% BSA at 20 ug/mL.

Techniques: Control

Journal: bioRxiv

Article Title: TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes

doi: 10.1101/2024.11.05.622187

Figure Lengend Snippet: ( A - B ) Five-day TGFβ2 treatment (1ng/mL) significantly altered expression of TGFβ pathway effectors, cytoskeletal machinery, and canonical fibrotic markers. ( C ) TGFβ2 treatment significantly increased TRPV4 and PIEZO1 expression, but not TREK1 and TRPC1 expression. Mean ± SEM shown. N = 4 - 8 experiments, each gene tested in 3-7 different pTM strains (See ). Two-tailed one sample t-test of TGFβ2-induced gene expression levels as a percent of control samples. ( D ) Isolation of membrane proteins from two separate pooled pTM samples suggests TGFβ2 treatment drives increased TRPV4 membrane insertion. N = 2 independent pooled samples, 3 pTM strains were pooled per sample. * P < 0.05, ** P < 0.01.

Article Snippet: Recombinant human TGFβ2 protein was ordered from R&D Systems and reconstituted in sterile 4 mM HCl with 0.1% BSA at 20 ug/mL.

Techniques: Expressing, Two Tailed Test, Control, Isolation, Membrane

Journal: bioRxiv

Article Title: TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes

doi: 10.1101/2024.11.05.622187

Figure Lengend Snippet: ( A ) Five-day TGFβ2 treatment (1 ng/mL) increased TRPV4 agonist-induced (GSK101, 10 nM) Ca 2+ influx in pTM cells compared to serum-free media alone treated cells tested on the same day (N = 5 pTM strains, n = 3 - 5 slides/condition/day, individual data points over mean ± SEM). Two-tailed one sample t- test of TGFβ2-treated cell average GSK101 response as a percent of control samples from the same pTM strain on the same day. ( B) Violin plots showing the distribution of GSK101-induced Ca 2+ responses for each pTM strain tested in A. Thick dashed line indicates mean, while light dashed line indicates quartiles. ( C ) Representative traces showing TRPV4 agonist-induced Ca 2+ influx (seen as an increase in F 340 /F 380 ) in pTM (mean ± SEM of 4 representative cells/ group), alongside example Fura-2-loaded pTM cells before (i), during (ii), and after (iii) GSK101 application. Scale bar = 50 µm. ** P < 0.01

Article Snippet: Recombinant human TGFβ2 protein was ordered from R&D Systems and reconstituted in sterile 4 mM HCl with 0.1% BSA at 20 ug/mL.

Techniques: Two Tailed Test, Control

Journal: bioRxiv

Article Title: TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes

doi: 10.1101/2024.11.05.622187

Figure Lengend Snippet: ( A ) Representative longitudinal 24-well plate scans of collagen type I hydrogels seeded with pTM subjected to the different treatments (dashed lines outline size of contracted constructs). ( B ) Longitudinal quantification of hydrogel construct size compared to the control group at the 0 minute time point. ( C ) Detailed comparisons between groups at each experimental time point. n = 6 hydrogels/group. One-way ANOVA with Tukey’s multiple comparisons test, data in ( B,D ) shows individual data points over mean ± S.D. One pTM strain shown: TGFβ2-induced contractility induction, HC-06-mediated rescue of hypercontractility, and GSK101-induced transient (15 min) contraction were consistent across (3/3) pTM strains tested (Figure S2). ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Recombinant human TGFβ2 protein was ordered from R&D Systems and reconstituted in sterile 4 mM HCl with 0.1% BSA at 20 ug/mL.

Techniques: Construct, Control

Journal: bioRxiv

Article Title: TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes

doi: 10.1101/2024.11.05.622187

Figure Lengend Snippet: ( A ) Intravitreal injection of LV-TGFβ2 (week 1), but not LV-Control, elevates IOP in WT mice (N = 5 eyes/group) as early as one-week post-injection. Injection of TRPV4 antagonist HC-06, but not PBS, produced multiday IOP reduction in LV-TGFβ2 treated eyes. HC-06 and PBS injections did not affect IOP in LV-Control injected eyes. Two-way ANOVA with Bonferroni post-hoc analysis ( B ) Direct comparison of the results of PBS and HC-06 injections in the eyes shown in A . Two-way ANOVA with Bonferroni post-hoc analysis ( C ) Intravitreal injection of LV-TGFβ2 in Trpv4 -/- mice (N = 6 eyes/group) resulted in only mild OHT; plotted against WT eyes at matching timepoints (3 WT cohorts including the 5 WT eyes shown in A-B, N = 8-15 eyes/group). ( D ) Statistical comparison of the IOP values shown in C. The IOP in LV-TGFβ2 WT eyes was significantly elevated compared to the LV-TGFβ2 Trpv4 -/- eyes from 2 weeks post-injection. LV-Control injected eyes in WT or Trpv4 -/- eyes remain close to the baseline value and are not significantly different. Two-way ANOVA with Bonferroni post-hoc analysis. ( A, C) shows mean ± SEM. Data in ( B, D ) shows individual data points over mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Recombinant human TGFβ2 protein was ordered from R&D Systems and reconstituted in sterile 4 mM HCl with 0.1% BSA at 20 ug/mL.

Techniques: Injection, Control, Produced, Comparison

Journal: bioRxiv

Article Title: TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes

doi: 10.1101/2024.11.05.622187

Figure Lengend Snippet: ( A ) ∼2 months post-LV injection daytime (12-2:00 P.M) and nocturnal (9-10:00 P.M.) IOP compared in WT mice (N = 4 eyes/group) before drug treatment. LV-TGFβ2 eyes were elevated at daytime, but nocturnal OHT was not significantly different between LV-Ctrl and LV-TGFβ2 eyes. One- way ANOVA with Tukey’s multiple comparisons test. ( B - C) PBS-injected eyes did not exhibit changes in daytime or nighttime intraocular pressure; however, HC-06 injection reduced TGFβ2-induced IOP elevations during the day and LV-Ctrl and LV-TGFβ2 nocturnal IOPs (N = 4 Eyes/Group); Two-way ANOVA with Bonferroni post-hoc analysis. Figures show datapoints over mean ± SEM, * P < 0.05 , ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Recombinant human TGFβ2 protein was ordered from R&D Systems and reconstituted in sterile 4 mM HCl with 0.1% BSA at 20 ug/mL.

Techniques: Injection

Journal: bioRxiv

Article Title: TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes

doi: 10.1101/2024.11.05.622187

Figure Lengend Snippet: Chronic exposure to TGFβ2 induces upregulation of functional TRPV4 channels alongside the autoinhibitory canonical modulator SMAD7. TRPV4-mediated Ca 2+ influx, canonical, and non-canonical TGFβ2 signaling stimulate Rho/ROCK signaling, augment cytoskeletal contractility, and stimulate ECM release to increase the flow resistance of the conventional pathway. Increased contractility drives OHT, resulting in a feedforward vicious TRPV4-dependent circle loop that maintains ocular hypertension. Schematic made using Biorender.com.

Article Snippet: Recombinant human TGFβ2 protein was ordered from R&D Systems and reconstituted in sterile 4 mM HCl with 0.1% BSA at 20 ug/mL.

Techniques: Functional Assay